# Covalent Inhibition as a Method to Counteract Botulinum Intoxication

> **NIH NIH R01** · SCRIPPS RESEARCH INSTITUTE, THE · 2021 · $637,866

## Abstract

PROJECT SUMMARY/ABSTRACT
Botulinum neurotoxin serotype A (BoNT/A), which causes the disease botulism, is the most potent toxin known
to man. BoNTs are most commonly encountered as BotoxTM, the increasing use of which has made iatrogenic
botulism a major concern. BoNTs are one of only six pathogens designated by the CDC as a category A
bioterrorism agent due to its toxicity and ease of production. Furthermore, the spread of botulism among heroin
users is a growing concern.
 Despite the potential threat and the severity of the disease, there is no therapeutic available for rescuing
the neuronal intoxication that causes botulism. At best, the progression of the disease is mitigated by treatment
with a heptavalent antitoxin, which still requires months of hospitalization. Our long-term goal is to develop a
clinically viable therapeutic capable of reversing the effects of botulinum neurotoxin, in addition to arresting
progress. As BoNT intoxication is a solitary event, we posit that an irreversible covalent inhibitor capable of
entering muscle neurons could permanently compromise its catalytic machinery, providing a solution to the
discrepancy between the lifetime of a small molecule in neurons and the persistence of the neurotoxin. In contrast
to the numerous reports of irreversible inhibitors of serine/cysteine proteases, irreversible inhibition of
metalloproteinases is rare, a result of differences in catalytic mechanisms. As such we have devised a strategy
wherein a covalent warhead that targets an allosteric reactive residue is tethered to a potent active site inhibitor,
thus creating a “bifunctional” inhibitor. This in essence skirts enzyme mechanistic issues and now allows covalent
targeting of the BoNT/LC.
 Based on promising preliminary data, we propose four specific aims that will lead to the identification of
potent, reactive, and selective molecules. 1) Using docking and structure activity relationship (SAR) data we will
adapt previously identified reversible inhibitors of BoNT/A light chain to the bifunctional covalent strategy. 2) We
will screen covalent fragments in the presence of reversible inhibitors to select for warheads that tolerate the
presence of the reversible inhibitor scaffold, and to account for active site conformational changes induced by
the reversible inhibitor. 3) We will obtain and analyze crystallographic, cell and pharmacokinetic data to iteratively
improve our inhibitors, prioritizing potency, selectivity, and safety in order to maximize the chance for success
during in vivo studies. 4) Finally, by testing our compounds in the FDA gold standard mouse lethality model, we
will assess the efficacy of our developed compounds and their suitability for pre-clinical and clinical studies.

## Key facts

- **NIH application ID:** 10177867
- **Project number:** 5R01AI153298-02
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Kim Janda
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $637,866
- **Award type:** 5
- **Project period:** 2020-06-02 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10177867

## Citation

> US National Institutes of Health, RePORTER application 10177867, Covalent Inhibition as a Method to Counteract Botulinum Intoxication (5R01AI153298-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10177867. Licensed CC0.

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